This invention relates to functionalised tetra-aza macrocycles, to metal complexes thereof, to conjugate compounds containing the functionalised tetra-aza macrocycles and metal complexes thereof and to their use in diagnosis and therapy.
The attachment of metal ions to proteins, peptides and other, smaller molecules is a fast expanding technology, which has numerous proven and potential applications in research, in industry and, particularly, in medicine.
In recent years, much of the impetus behind the development of this technology has been the ability to link metal ions to antibodies, especially monoclonal antibodies. Such metal labelled antibodies have found a widespread use, especially in medicine, where they have been employed, for example, to target the metal ion to a specific tissue type, both in vitro and in vivo. Thus, metal labelled antibodies have applications in locating specific tissue types (e.g. employing computer-aided tomographic techniques where the metal ion is in some way detectable) and in the treatment of cell disorders (e.g treating mammalian tumours where the metal ion is a cytotoxic radionuclide).
Conventionally, attachment of the metal ion to a protein such as an antibody has been achieved by complexation by an acyclic chelate such as a substituted diethylenetriaminepentaacetic acid [Gansow O. A. et al, Inorg. Chem., (1986), 25, 2772] or ethylenediaminetetraacetic acid [Meares, C. F. et al, Acc. Chem. Res., (1984), 17, 202] covalently linked to the antibody. Such acyclic complexes however tend to be unstable in vivo either as a result of acid-catalysed decomplexation or competitive chelate binding by Ca2+ or Zn2+ in serum, or as a result of competition from transferrin [Moerlein, S. M. et al, Int. J. Nuc. Med. Biol., (1981) 8, 277]. The lack of stability can result in uncomplexed metal atoms in the body which have a cytotoxic effect on healthy tissue (e.g. bone marrow) or which markedly reduce the signal-to-noise ratio of an imaging technique.
A possible alternative to the use of acyclic chelates in the labelling of antibodies is the use of macrocyclic ligands, which has been suggested in broad terms [Gansow O. A. et al. Am. Chem. Soc. Symp. Ser., (1984), 241, 215; UK Patent Specification Publication No. 2122641; and Moi M. K. et al, Anal. Biochem., (1985), 148, 249-253].
We have now found a new class of functionalised tetra-aza macrocyles, members of which are able to form more kinetically inert complexes with metal ions than are chelating agents conventionally in use for the attachment of metal ions to proteins and other molecules. The macrocycles of the invention are particularly useful for attachment to proteins, especially antibodies, to provide conjugate compounds capable of binding metals to give complexes which are advantageously stable in vivo.
Thus, according to one aspect of the present invention we provide a compound of general formula (1): 
wherein
m and n, which may be the same or different, is each zero or an integer 1, 2, or 3;
d is zero or an integer 1, 2 or 3;
q is zero or an integer from 1 to 6 inclusive;
R, R1, R2 and R3, which may be the same or different, is each a hydrogen atom or an alkyl, alkoxyalkyl, xe2x80x94CO2H, xe2x80x94SO3H, xe2x80x94PO3H2 or aryl group;
L is a covalent bond or a linker group;
Z is a hydrogen atom or a reactive functional group, with the proviso that when L is a covalent bond Z is a reactive functional group;
and metal complexes and/or salts thereof.
In the compounds of formula (1), alkyl groups represented by R, R1, R2 and R3 may be for example C1-6alkyl groups such as methyl or ethyl groups. Alkoxyalkyl groups represented by R, R1, R2 or R3 may be for example C1-3alkoxyC1-3alkyl groups e.g. methoxymethyl. When R, R1, R2 or R3 is an aryl group it may be for example a substituted phenyl group, such as a group of formula 
(where R4 is a hydrogen atom or a C1-6alkyl, e.g. methyl, C1-3alkoxy1-3alkyl, e.g. methoxymethyl, or C6-12aryl, e.g. phenyl group).
In general, compounds of formula (1) in which R, R1, R2 and R3 are the same are preferred. Compounds of this type in which q is an integer from 1 to 6 inclusive, particularly an integer 1, and R1, R2 and R3 are xe2x80x94SO3H, xe2x80x94PO3H2
or, in particular, xe2x80x94CO2H, are especially preferred.
In the compounds of formula (1), it will be appreciated that the nature of the group L when it is a linker group may be varied widely without substantially affecting the usefulness of compounds of formula (1) and the metal complexes thereof. Thus L may be any suitable organic radical and may be for example an optionally substituted aliphatic hydrocarbyl chain, optionally interrupted by one or more heteroatoms selected from xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 or by one or more xe2x80x94N(R5)xe2x80x94 (where R5 is a hydrogen atom or a C1-6alkyl group), xe2x80x94CON(R5)xe2x80x94, xe2x80x94N(R5)COxe2x80x94, cycloalkylene, aromatic, or heteroaromatic groups.
In the above definition, and in the same context whenever it appears below, the term xe2x80x9cinterrupted byxe2x80x9d as applied to cycloaliphatic or aromatic groups is to be understood to also mean that these particular groups may additionally be present linked to the terminal carbon atom of the hydrocarbyl chain represented by L, at the opposite end of the chain to the carbon atom attached to the macrocycle.
Thus, for example, L may be an optionally substituted straight or branched C1-20alkylene, C2-20alkenylene, or C2-20alkynylene chain, optionally interrupted by one or more xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 atoms or C5-8cycloalkylene (e.g. cyclopentylene or cyclohexylene), C6-12aromatic (e.g phenylene or substituted phenylene), C5-10heteroaromatic (e.g furanyl, pyridyl), xe2x80x94N(R5)xe2x80x94, xe2x80x94CON(R5)xe2x80x94 or xe2x80x94N(R)5COxe2x80x94 groups.
Examples of substituents which may be present on the chain L include halogen atoms, e.g. fluorine, chlorine, bromine, or iodine atoms or groups selected from C1-6alkoxy (e.g methoxy or ethoxy), hydroxy, nitro, xe2x80x94N(R6)(R7), [where R6 is a hydrogen atoms or a C1-6alkyl group and R7 is a C1-6alkyl group; e.g. xe2x80x94NHCH3 or xe2x80x94N(CH3)2], or substituted amido, e.g. a group of formula xe2x80x94(CH2)nCON(R8)(R9) [where n is zero or an integer 1 to 4 inclusive, R8 is a hydrogen atom or a C1-6alkyl group, e.g. methyl and R9 is an optionally substituted C1-6alkyl group].
Substituted alkyl groups represented by R9 include for example C1-6alkyl groups substituted by one or more halogen atoms, or nitro, amino or hydroxy groups.
In general, in compounds of formula (1) the linker group is preferably an optionally substituted C1-10alkylene, (especially C1-6alkylene such as methylene, ethylene, propylene butylene, pentylene or hexylene) C2-10alkenylene or C2-10alkynylene chain optionally interrupted by one or more xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94 atoms or cyclohexylene, phenylene, substituted phenylene, xe2x80x94NHxe2x80x94, xe2x80x94N(CH3)xe2x80x94, xe2x80x94CONHxe2x80x94, xe2x80x94CON(CH3)xe2x80x94 xe2x80x94NHCOxe2x80x94 or xe2x80x94N(CH3)COxe2x80x94 groups.
Particular examples of linker groups represented by L include, for example, xe2x80x94(CH2)dxe2x80x94 (where d is an integer 1 to 4 inclusive), 
xe2x80x94(CH2)dNHCO(CH2)exe2x80x94 (where e is an integer 1 to 4 inclusive) and xe2x80x94(CH2)dNHCO(CH2)eOCH2xe2x80x94.
The reactive functional group represented by Z in compounds of formula (1) may be any group capable of reacting with a thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group. Aromatic groups include, for example, phenolic groups. Heteroaromatic groups include for example imidazolyl groups.
Thus, Z may be, for example,
(i) a halogen atom (as for example a chlorine, bromine or iodine atom),
(ii) a group selected from xe2x80x94SH, xe2x80x94NH2, hydrazino (xe2x80x94NHNH2) or a derivative thereof, (for example xe2x80x94N(CH3)NH2, xe2x80x94NHCONHNH2, xe2x80x94NHCSNHNH2, or phenyl hydrazino),
(iii) xe2x80x94NCO, xe2x80x94NCS, xe2x80x94COR10 in which R10 is a halogen atom (as for example a chlorine or bromine atom), N3, C1-6alkoxy, (as for example methoxy), C6-12aryloxy (as for example nitrophenyloxy or dinitrophenyloxy), imidyloxy (as for example succinimidyloxy) or imidazolyloxy,
(iv) imido, (as for example maleimido), or
(v) a vinyl group of the formula xe2x80x94Het1xe2x80x94C(R12)xe2x95x90CH2 in which
Het1 and R12, which may be the same or different, are each a nitrogen containing heterocyclic group, (as for example a pyridyl group) or
Het1 is a nitrogen containing heterocyclic group and R12 is a hydrogen atom
as for example a vinyl pyridyl group of formula: 
(where R11 is a C1-4alkyl e.g. methyl, group).
Metal complexes of the compounds of formula (1) include complexes wherein the metal is di- or tripositive and has a coordination number 6 or greater, especially 8. Examples of such metals include indium (In), copper (Cu), lead (Pb), bismuth (Bi), yttrium (Y), terbium (Tb), gadolinium (Gd) and Scandium (Sc). Y, Pb, Tb, Gd, and Sc are preferred, particularly Y, Gd and Pb. In general the metal is preferably a radioactive isotope. Yttrium, especially 90Y, is particularly preferred.
In general, optimum binding of the metal to the compounds of formula (1) may be achieved by selection of the ring size and where appropriate by adjusting the potential coordination number by choice of the group (CH2)qR, xe2x80x94(CH2)qR1, xe2x80x94(CH2)qR2, and/or xe2x80x94(CH2)qR3 Thus a particularly important class of compounds of formula (1) is that wherein d is an integer 1. Especially useful compounds are those wherein d is an integer 1, m is an integer 1 or 2 and n is an integer 1 or 2. In general, compounds of formula (1) in which xe2x80x94(CH2)qR, xe2x80x94(CH2)qR1, xe2x80x94(CH2)qR2 and xe2x80x94(CH2)qR3 is each xe2x80x94CH2CO2H are particularly useful.
Salts of the compounds of formula (1) include salts with bases, e.g. sodium or potassium salts, or acid addition salts such as hydrobromides or hydrochlorides. Pharmaceutically acceptable salts are particularly preferred.
A particularly useful group of compounds of the invention has the formula (1) wherein R, R1, R2, R3, m, n, d ,and q are as defined for formula (1) and the groups xe2x80x94L and Z together represent a group
xe2x80x94(CH2)rxe2x80x94Xxe2x80x94Y in whichxe2x80x83xe2x80x83(1)
r is zero or an integer from 1 to 6 inclusive,
X is:
xe2x80x94NHxe2x80x94, 
xe2x80x83in which R4 is as previously defined and s is zero or an integer from 1 to 4 inclusively,
xe2x80x94(CH2)sNHNHxe2x80x94, or
xe2x80x94(OCH2CH2)tNHxe2x80x94 in which t is an integer from 1 to 6 inclusively, and
Y is a group xe2x80x94COZ1 or xe2x80x94CO(R)Z1 in which
R is a spacer group, and
Z1 is a group xe2x80x94(CH2)tHal in which Hal is a halogen atom; 
xe2x80x83where R12 is hydrogen or a nitrogen containing aromatic heterocyclic group, as for example a pyridyl group, or
xe2x80x94(CH2)rNCS;xe2x80x83xe2x80x83(2)
and the metal complexes and/or salts thereof.
In compounds of this type, the spacer group R may be for example an alkylene, e.g. ethylene, alkoxyalkylene, e.g. methoxymethylene, aryl, e.g. phenylene, aralkylene, e.g. phenalkylene such as phenethylene, or cycloalkylalkylene, e.g. cyclohexylmethylene group.
A further particularly useful group of compounds according to the invention has the formula (1) wherein R, R1, R2, R3, m, n, d and q are as defined for formula (1) and the groups L and Z together represent a group xe2x80x94(CH2)rXH (where r and X are as defined above) and the metal complexes and/or salts thereof.
An important group of compounds according to the invention has the formula (1a): 
wherein R, R1, R2, R3, L and Z are as defined for formula (1) and metal complexes ad/or salts thereof.
Compounds of this type in which R, R1, R2 and R3 is each xe2x80x94CO2H are particularly preferred.
Compounds of formula (1a) in which L is a linker group [particularly those specifically identified for compounds of formula (1)] are especially useful.
Z in compounds of formula (1a) is preferably a reactive functional group, particularly those specifically identified for compounds of formula (1), especially a group of formula xe2x80x94Het1xe2x80x94CR12xe2x95x90CH or a dione of formula 
Yttrium complexes of the compounds of formula (1a) are particularly useful.
Yttrium complexes of the compounds of formula (1a) are particularly useful.
The compounds of formula (1) and the metal complexes and/or salts thereof have a diagnostic use as imaging agents in vitro and in vivo. The compounds of formula (1) and the metal complexes and/or salts thereof are also cytotoxic agents and may be used in the treatment of abnormal cell disorders, for example in the treatment of tumours.
For application of the compounds of formula (1) as imaging or cytotoxic agents, it is generally preferable to couple the compounds to other molecules such as proteins, especially antibodies, peptides or carbohydrates to form conjugate compounds, and the compounds of formula (1) are particularly well adapted for use in this respect.
Thus, according to a further aspect of the invention, we provide a conjugate compound which comprises a compound of formula (1), or a metal complex and/or salt thereof, coupled to a protein, peptide or carbohydrate.
The compound of formula (1) may be coupled through any thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group present in the protein, peptide or carbohydrate.
In a preferred aspect of the invention, we provide a conjugate compound which comprises a compound of formula (1) or a metal complex and/or salt thereof, coupled to an antibody.
It is to be understood that conjugate compound according to the invention may contain more than one molecule of a compound of formula (1) coupled to any one protein, peptide or carbohydrate molecule.
In a particular aspect, the invention provides a conjugate compound of formula 
wherein m, n, d, q, R, R1, R2, R3, and L are as defined for formula (1);
Z1 is the residue of a reactive functional group;
w is zero or an integer 1;
z is an integer 1 or more;
Ab is an antibody; and metal complexes and/or salts thereof.
In the compounds of formula (2), the residue of a reactive functional group represented by Z1 may in general be the residue of a reactive functional group Z as defined for formula (1).
In particular, Z1 may be for example xe2x80x94Sxe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94NHNxe2x95x90, N(CH3)Nxe2x95x90, xe2x80x94NHCONHNxe2x95x90, xe2x80x94NHCSNHNxe2x95x90, xe2x80x94N(Ph)Nxe2x95x90 in which Ph is phenyl, xe2x80x94NC(O)xe2x80x94, xe2x80x94NC(S)xe2x80x94, xe2x80x94COxe2x80x94, 
xe2x80x94Het1xe2x80x94C(R12)CH2xe2x80x94 or 
The antibody Ab in the conjugates of formula (2) may be a complete antibody molecule or a fragment thereof, or an analogue or either of these, provided that the antibody comprises of a specific binding region. Thus the antibody may be polyclonal, or, preferably, monoclonal, or a fragment thereof for example a Fabxe2x80x2 or F(ab)2xe2x80x2 fragment. If desired the antibody may be a recombinant antibody, (i.e. an antibody which has been produced using recombinant DNA techniques). The antibody may be a chimaeric antibody comprising linked antibody fragments, each from a different source (see for example International Patent Specification No. WO 86/01533).
The antibody may be specific for any number of antigenic determinants, but is preferably specific for one antigenic determinant. Particular determinants include tumour cell-associated antigens, particularly mammalian tumour cell antigens for example oncofetal antigens such as carcinoembryonic antigen or alphafetoprotein.
A particular useful antibody is that known as B72.3 [Colcher, D. et al Proc. Nat. Acad. Sci. USA (1981), 78, 3199].
The antibody Ab will in general be coupled to the remainder of the conjugate of formula (2) (i.e. the macrocycle and linker) through any appropriate reactive atom or group, for example a nitrogen or, especially, sulphur atom, present in the antibody. It will be appreciated that any one antibody molecule may contain more than one reactive group capable of coupling with the macrocycle and linker. Thus, for example, z in the conjugates of formula (2) may be an integer 1, 2, 3, 4, 5, 6 or more depending on the number of macrocycles linked to any particular antibody molecule or fragment or analogue thereof.
Yttrium complexes of conjugates of formula (2) are particularly useful.
It is to be understood that the definitions and preferences expressed for m, n, d, q, R, R1, R2, R3 and L in compounds of formula (1), and for classes of compounds of formula (1) are also applicable to conjugates of formula (2).
Particularly useful conjugate compounds according to the invention are those comprising a compound of formula (1a), or a metal complex and/or salt thereof, coupled to an antibody. The indium complexes of these conjugates are especially important.
The compounds of formulae (1) and (2) may be formulated for use in accordance with conventional practice, and thus according to a further aspect of the invention we provide a composition comprising a compound of formula (1) or a compound of formula (2) or a metal complex and/or salt thereof, together with one or more pharmaceutically acceptable carriers.
Particularly suitable compositions according to the invention are those adapted for parenteral administration, especially intravenous administration. Suitable formulations of this type include solutions of the compounds of formulae (1) or (2) in isotonic saline.
The quantities of compounds of formulae (1) or (2) used in formulations according to the invention will vary according to the intended use (i.e. imaging or therapy) and other variables such as the intended cell target, but may be easily determined in accordance with conventional practice for reagents of this type.
Compounds of the invention may be prepared by the following processes wherein the groups and symbols R, R1, R2, R3, m, n, d, q, L, Z, Ab and z are as defined for formulae (1) and (2) except where stated otherwise. Where a metal complex is desired as a final product, the complexation with a metal atom may be carried out as a final step in the production process, as described below for the complexation of compounds of formulae (1), or alternatively it may be desirable to complex the metal at an earlier stage in the process, providing of course that the requisite macrocycle structure is present. In the following processes, it may be desirable to use starting materials in which the group Z is in a protected state, or which contain a precursor of the group, as discussed below.
Thus, according to a further aspect of the invention a compound of formula (1) or a metal complex thereof may be prepared by reaction of a corresponding compound of formula (3) 
or a metal complex thereof, with a reagent R1(CH2)qD (where D is a displaceable group). Displaceable groups represented by D include for example halogen atoms, for example a bromine, chlorine or iodine atom.
The reaction may be performed in a solvent such as water or an organic solvent such as a nitrile e.g. acetonitrile or an alcohol e.g. isopropanol or an amide e.g. dimethylformamide in the presence of a base, e.g. an inorganic base such as an alkali metal carbonate or hydroxide, e.g. sodium, potassium or caesium carbonate, or sodium, potassium or lithium hydroxide, at a high temperature e.g. the reflux temperature.
In this reaction, the group Z may need to be in a protected state. Conventional protecting groups may be used, depending on the nature of Z, and may be removed using standard procedures, once the desired reaction has been effected. Similarly, when the reagent R1(CH2)qD contains an acid group this may also need to be protected, for example as an ester e.g. a methyl ester. The acid may be re-generated after the desired reaction is complete, for example by hydrolysis using an acid such as sulphuric acid.
It will be appreciated that where it is desired to prepare a compound of formula (1) in which R, R1, R2 and R3 are not the same this may be achieved by first selectively N-protecting the compound of formula (3) or a precursor using an appropriate amine protecting group(s), for example a p-toluenesulphonyl group as described below, in accordance with conventional practice. Reaction of the N-protected compound (3) with R1(CH2)qD followed by deprotection and further reaction as necessary with other reagents R1(CH2)qD then yields the desired compound in which R, R1, R2 and R3 are not the same.
Where metal complexes of compounds of formulae (1) or (2) are required (or any other suitable macrocyclic intermediate described herein) these may be prepared by treating the compound with a metal salt (for example a metal halide) in an appropriate solvent for example an aqueous or non aqueous solvent, (e.g. acetonitrile, acetone, propylene carbonate, dimethylformamide or dimethylsulphoxide) at any suitable temperature from 0xc2x0 C. to 100xc2x0 C. such as 10xc2x0 to 80xc2x0 C. e.g. around 60xc2x0 C.
In another process, a compound of formula (1) or a metal complex thereof wherein R, R1, R2 and R3 is each xe2x80x94(CH2)qPO3H2 (where q is an integer 1 to 6) may be prepared by reaction of a compound of formula (3) or a metal complex thereof with phosphorous acid and an aldehyde RbCHO (where Rb is a hydrogen atom or a C1-5alkyl group) in the presence of an acid, such as hydrochloric acid at an elevated temperature, e.g. 100xc2x0-130xc2x0 C.
Compounds of formula (1) may also be prepared by interconversion from other compounds of formula (1). Thus one functional group Z may be exchanged for another and, if desired a linker group L changed to another by appropriate manipulative reactions. For example, a compound of formula (1) where xe2x80x94Lxe2x80x94Z is a group xe2x80x94L1xe2x80x94NHCOxe2x80x94L2xe2x80x94Z (where xe2x80x94L1xe2x80x94NHCOxe2x80x94L2 represents the group L) may be prepared by reaction of a corresponding compound wherein xe2x80x94Lxe2x80x94Z represents xe2x80x94L1xe2x80x94NH2 with a reagent RbOxe2x80x94L2xe2x80x94Z (where Rb is for example an imide, such as succinimide, or a substituted phenyl group such as a p-nitrophenyl group) in the presence of a tertiary amine, such as diisopropylethylamine, in a solvent such as dimethylformamide.
Reagents of formula RbOxe2x80x94L2xe2x80x94Z are either known compounds or may be obtained form known starting materials using methods analogous to those used for the preparation of the known compounds.
A conjugate compound of formula (2) or a metal complex thereof may be prepared by reaction of a corresponding compound of formula (1) or a metal complex thereof with an antibody Ab (as previously defined).
The reaction may be performed in a suitable solvent, for example an aqueous solvent such as a phosphate buffer, at an appropriate temperature, for example at 0xc2x0-30xc2x0 C., especially 0xc2x0-10xc2x0 C. e.g. 4xc2x0 C.
The antibody Ab may be obtained using procedures well known in the art. If desired, before the coupling reaction, the antibody may first be treated to yield appropriate groups for reaction with the compound of formula (1). Thus for example the antibody may be subjected to oxidation, for example periodate oxidation to yield aldehyde groups, or, in particular, may be treated with a reagent [e.g. Traut""s reagent (2-iminothiolane)] using standard procedures to generate free sulphydryl groups in the molecule.
Salts of compounds of formulae (1) or (2) and their metal complexes may be prepared by conventional means, for example by reaction with an appropriate base or acid in a suitable aqueous solvent.
Intermediates of formula (3) may be prepared by deprotection of a compound of formula (4) 
(where R13 is a protecting group such as a p-toluenesulphonyl group. The deprotection will depend on the nature of the protecting group R13. Thus, for example, when R13 is a p-toluenesulphonyl group removal of this may be achieved by treatment of the compound of formula (4) with an acid, for example HBr-acetic acid, in the presence of phenol at a high temperature, or by reaction with lithium in liquid ammonia in the presence of an alcohol such as ethanol.
Intermediates of formula (4) may be prepared by treating a compound of formula (5)
R13NH(CH2)nCH2N(R13)CH2CH(Lxe2x80x94Z)N(R13)CH2(CH2)mNHR13
with a compound R13OCH2(CH2)dOR13 in the presence of a base such as sodium ethoxide in a solvent such as dimethylformamide.
Intermediates of formula (5) may be prepared by reaction of compounds of formula (6)
xe2x80x83R13NH(CH2)nCH2NHCH2CH(Lxe2x80x94Z)NHCH2(CH2)mNHR13xe2x80x83xe2x80x83(6)
with a protecting agent, for example p-toluenesulphonyl chloride in a base such as pyridine.
Intermediates of formula (6) in which m and n are the same may be prepared by reaction of a diamine of formula (7):
H2NCH(Lxe2x80x94Z)CH2NH2xe2x80x83xe2x80x83(7)
with a reagent R13NH(CH2)mCOHal (where Hal is a halogen atom) in the presence of a base such as triethylamine, followed by reduction using for example borane in a solvent such as tetrahydrofuran at a high temperature e.g. the reflux temperature, followed by treatment with an acid such as hydrochloric acid.
Where it is desired to prepare an intermediate of formula (9) in which m and n are not the same a protected amine H2NCH(Lxe2x80x94Z)CH2NHR13 may be used in the above reaction. Removal of the protecting group after the reaction followed by repeated alkylation with a different compound R13NH(CH2)nCOHal then yields the required intermediate.
Diamines of formula (7) may be prepared from an appropriately substituted amino acid of formula (8):
H2NCH(Lxe2x80x94Z)CO2R11xe2x80x83xe2x80x83(8)
by reaction with ammonia in a solvent such as methanol, followed by reduction using for example lithium aluminium hydride.
The substituted amino acids of formula (8) are either known compounds or may be prepared using methods analogous to those used for the preparation of the known compounds.
In an alternative process, intermediates of formula (5) may be prepared by reaction of a compound of formula (9)
R13HNCH(Lxe2x80x94Z)CH2N(R13)CH2(CH2)nNHR13xe2x80x83xe2x80x83(9)
with a compound R13OCH2(CH2)dN(R13)(CH2)mCH2OR13 in the presence of a base such as caesium carbonate in a solvent such as dimethylformamide.
Intermediates of formula (9) may be prepared by reduction of compounds of formula (10)
H2NCH(Lxe2x80x94Z)CONHCH2(CH2)nNHR13xe2x80x83xe2x80x83(10)
using for example borane as described above, followed by reaction to introduce the protecting group R13, for example with p-toluenesulphonyl chloride in a solvent such as dichloromethane in the presence of a base such as triethylamine at e.g. reflux.
Intermediates of formula (10) may be prepared by reaction of an appropriately substituted amino acid of formula (8) (where R11 is a methyl or ethyl group) with a diamine H2NCH2(CH2)nNH2 at a high temperature, e.g. the reflux temperature.
The invention is illustrated by the following Examples.